“L23 Protein Functions as a Chaperone Docking Site on the Ribosome”

1. “L23 Protein Functions as a Chaperone Docking Site on the Ribosome” Kramer, G., et. al. (2002) Nature419 171-174 Presented by Michael Evans Department of Chemistry and Biochemistry University of Notre Dame Notre Dame, IN 46616

2. Overview • Introduction to chaperones • Experiments and Results • Conclusions • Future Work

3. Chaperones and Folding • Newly synthesized polypeptides must fold to native conformation in crowded environment of the cell • Chaperones help many to avoid aggregation • Bind to exposed hydrophobic regions • PPIase activity • ATP dependent binding • Maintain conformational flexibility

4. Chaperone Pathway in Bacteria Hartl, F.U. and Hayer-Hartl, M. (2002) Science295 1852-1858

5. Trigger Factor (TF) • First bacterial chaperone to see nascent polypeptide • Has PPIase activity, but recognizes hydrophobic residues • Function overlaps with DnaJ/DnaK chaperones • N-terminal domain mediates binding to 50S subunit of ribosome

6. Significance • Explain coupling of synthesis to folding • Eukaryotic parallels • No TF • Other chaperones interact with ribosome • SRP study

7. A Few Questions • What part of TF is important for interaction with the ribosome? • Which ribosomal protein(s) and/or RNA does TF interact with? • Must TF bind ribosomes to interact with nascent chains? • Is ribosomal association required for TF’s participation in protein folding?

8. TF Signature • Alignment of TF homologues revealed 17 conserved residues • Completely conserved G-F-R-X-G-X-X-P motif--the TF signature • TF signature located in unstructured region • Could be surface-exposed and contribute to ribosome interaction

9. TF Signature and Mutants

10. TF Signature Mutants • FRK/AAA: should show reduced association with ribosomes

11. FRK/AAA Mutant Association with Ribosomes • Incubated FRK/AAA with ribosomes from tigE. coli • Ribosomes separated from unbound protein by centrifugation • SDS-PAGE of pellet (ribosome) and supernatant (unbound protein)

12. FRK/AAA Mutant Association with Ribosomes • Increased amount of FRK/AAA in supernatant relative to wt TF incubated with ribosomes S: Supernatant P: Ribosome Pellet

13. TF Signature Mutants • D42C: replace Asp with Cys to allow attachment of crosslinking reagent • BPIA is UV activatable • Attacks C-H bonds, so will react with ribosomal proteins and RNA

14. D42C Mutant Association and Crosslinking with Ribosomes • Couple TF D42C to BPIA • Incubate with tig ribosomes • Activate BPIA by UV irradiation • Separate ribosome-protein complexes as before by centrifugation • SDS-PAGE to resolve crosslinking products

15. D42C Mutant Association and Crosslinking with Ribosomes • Two products, 68 kDa and 75 kDa • RNase A treatment does not affect mobility of products • Trypsin digestion followed by ESI-MS to identify cross-linked proteins • 68 kDa: TF + L29 • 75kDa: TF + L23

16. Interaction is Specific • Add 2.5 M excess of either wt TF or FRK/AAA to compete with D42C-BPIA during crosslinking • wt TF results in decrease of both crosslinking products • FRK/AAA does not decrease yield of crosslinking products • Crosslinking products are a result of a specific TF-ribosome interaction

17. L23 and L29 • Both proteins of the large subunit • In direct contact with each other • Located next to the exit tunnel • Does TF associate directly with one or both?

18. L23 and L29 Deletion Mutants Strategy: replace ORF with kanamycin resistance cassette Adapted from Datsenko, K.A., and Wanner, B.L. (2000) Proc. Nat. Acad. Sci.97 6640-6645

19. L23 and L29 Deletion Mutants • Two mutants produced: • rpmC::kan, deletion of L29 gene • rplW::kan, deletion of L23 gene • rpmC::kan grows, but slightly slower than wt • rplW::kan requires presence of pL23 for growth

20. L23 and L29 Deletion Mutants • rplW::kan growth dependent on IPTG induction of pL23 • L23 mutant is also viable

21. L29 and TF Binding • Purify ribosomes from rpmC::kan under high salt conditions • Does TF remain bound to ribosomes without L29? • Can TF rebind ribosomes without L29?

22. TF Remains Associated to L29-Deficient Ribosomes • SDS-PAGE of isolated ribosomes • Control is from rplW cells with wt L23 from plasmid • TF remains associated with L29-deficient ribosomes

23. TF Can Rebind to L29-Deficient Ribosomes • SDS-PAGE of ribosome-TF pellet and supernatant • Control is from rplW cells with wt L23 from plasmid • TF associates with L29-deficient ribosomes

24. L23 Deletion and Mutants • L29 is not required for TF binding, but what about L23? • rplW mutants are nonviable, but pL23 rescues • What part of L23 is important for binding?

25. L23 Region 1 and 2 Mutants • Criteria for interaction: • residue is surface-exposed • Conserved among bacterial L23s • Two regions identified

26. L23 Region 1 and 2 Mutants • Region 1: E18A, E18Q, VSE/AAA • Region 2: E52K, FEV/AAA • All mutant L23s complement rplW

27. L23 Mutants and TF Binding • Only region 1 mutants have effect on TF binding • Does TF remain associated with ribosomes containing mutant L23? • Can TF rebind ribosomes containing mutant L23?

28. L23 Mutants and TF Binding • SDS-PAGE of isolated ribosomes • Control is from rplW cells with wt L23 from plasmid • TF does not remain associated with mutant L23 ribosomes

29. L23 Mutants and TF Binding • SDS-PAGE of ribosome-TF pellet and supernatant • Control is from rplW cells with wt L23 from plasmid • Little TF binds to mutant L23 ribosomes

30. L23 Mutants and TF Binding • Less TF co-purifies with ribosomes under physiological salt concentrations • Mutant L23 levels are consistent with wt ribosomal proteins

31. TF Interacts Directly with L23 • Create S-tagged L23-thioredoxin fusion (Trx-L23) • Bind to S-tag column and apply TF or FRK/AAA • Elute bound proteins

32. TF Interacts Directly with L23 • TF binds L23, but FRK/AAA binding is weak • TF and FRK/AAA have similar substrate binding properties • L23-TF interaction is not mediated through nascent polypeptide

33. TF • Nascent Polypeptide Interaction and L23 • Must TF bind L23 to interact with nascent polypeptide? • Use in vitro transcription/translation (IVT) and crosslinking • Produce 35S-labeled isocitrate dehydrogenase (ICDH) fragment • Use crosslinker to probe for TF-ICDH interaction

34. In Vitro Transcription/ Translation System • Translation competent fraction from tigE. coli • Purified ribosomes with wt L23, region 1 L23 mutants, or no L29 • Purified TF • Produce N-terminal fragment of ICDH, an in vivo TF substrate

35. Crosslinking • Crosslinker is disuccinimidyl suberate (DSS) • Homobifunctional • Spans 11.4 angstroms • Reacts with -amino groups of Lys to give crosslink and N-hydroxy succinimide (NHS) DSS NHS

36. Identifying Crosslink Results • Immunoprecipitate crosslink product with anti-TF Ab • IP and non-IP samples examined by elecrophoresis, autoradiography • Control with no DSS

37. L23 is Required for TF • ICDH Interaction • wt L23 yields strong TF-ICDH crosslinks • L23 mutants retard crosslinking • Co-IP w/anti-TF Abs confirms identity • Glu 18 mutants reduce TF-ICDH interaction

38. TF-Ribosome Interaction and In Vivo Protein Folding • Combine rplW::kan with dnaK • Compensate with plasmids for wt or mutant L23 • Examine growth and aggregation at different temperatures

39. TF-Ribosome Interaction and in vivo Protein Folding • wt L23 compensates for deletion • L23 mutations lethal at 37ºC

40. TF-Ribosome Interaction and in vivo Protein Folding • Aggregates isolated from double mutants • Aggregation increases with temperature • VSE/AAA mutation is most severe

41. The Big Picture

42. Conclusions • L23 is the TF docking site on the ribosome • Glu 18 is critical for binding • Mutations in TF or L23 which inhibit binding affect protein folding, growth • L23 couples protein synthesis with chaperone-assisted folding

43. Future Directions • Why does TF form two crosslinks to nascent chains? • What is the nature of the L23-TF binding interface? • Does temp increase rate of aggregation or TF-L23 on-off rate? • Role for eukaryotic L23 in recruiting chaperones?